Fig. 1: Generation of LmexCen^−/− null mutant with no antibiotic selection marker activity.

a Scheme of the Leishmania CRISPR vector pLdCN a&b expressing the Cas9 nuclease and two gRNAs targeting the 5′ and 3′ flanking sequences of L. mexicana centrin gene. b The CRISPR strategy to delete L. mexicana centrin gene. The small filled black rectangle in front of Bleomycin resistance gene (Ble) represents a 92 bp pyrimidine track which is required for trans-splicing processing of Ble transcripts. c PCR analysis with primer pairs F1R1 and F2R2 verified that both alleles of L. mexicana centrin gene were deleted as planned. d The CRISPR strategy to partially delete and inactivate the Ble gene targeted in the centrin gene locus. e DNA sequencing revealed that 160 bp of Ble gene sequence has been deleted after CRISPR double gRNA targeting and oligo donor transfection. CRISPR1 first round of CRISPR targeting, CRISPR2 second round of targeting. The sequences in green are the partial remaining or the complete gRNA targeting sites. The start codon ATG and stop codon TGA for Ble gene are highlighted in Red. f Amino acids alignment shows the remaining Ble sequence in the centrin gene locus is only able to encode 9 amino acids identical to the wild-type Ble gene product; the 160 bp DNA sequence deletion also caused a frame shift resulting in non-mature stop codon. Dashed lines represent the remaining sequence to the WT Ble gene; Dot lines represent the deleted sequences. g Sequencing coverage across each of the five centrin gene family members and partial flanking UTRs in the L. mexicana gene edited strain. The CRISPR targeted LmxM.22.1410 gene coverage is shown in red, non-targeted centrin genes are shown in shades of blue. h Percent coverage (y-axis) of all genes across the L. mexicana genome (x-axis) is shown in blue. The location of the centrin gene family members is highlighted by a dotted line and the genes are colored in red.